Study of plasmid mediated quinolone resistance genes among Escherichia coli and Klebsiella pneumoniae isolated from pediatric patients with sepsis

The resistance to antibiotics in Gram-negative bacilli causing sepsis is a warning sign of failure of therapy. Klebsiella pneumoniae (K. pneumoniae) and Escherichia coli (E. coli) represent major Gram-negative bacilli associated with sepsis. Quinolone resistance is an emerging resistance among E. coli and K. pneumoniae. Therefore, the present study aimed to study the presence of plasmid-mediated quinolone resistance (PMQR) genes qnrA, qnrB, and qnrS by polymerase chain reaction (PCR) in E. coli and K. pneumoniae isolated from pediatric patients with sepsis. This was a retrospective cross-sectional study that included pediatric patients with healthcare-associated sepsis. The E. coli and K. pneumoniae isolates were identified by microbiological methods. PMQR genes namely qnrA, qnrB, and qnrS were detected in E. coli and K. pneumoniae isolates by PCR. The results were analyzed by SPPS24, and the qualitative data was analyzed as numbers and percentages and comparison was performed by Chi-square test, P was significant if < 0.05. The most prevalent gene detected by PCR was qnrA (75%), followed by qnrB (28.1%), and qnrS (25%). The most frequently detected qnr gene in E coli and K. pneumoniae was qnrA (28.8%, and 16.3% respectively). The present study highlights the high prevalence of ciprofloxacin resistance among E. coli and K. pneumoniae isolated from pediatric patients with healthcare-associated sepsis. There was a high frequency of PMQR genes in E. coli and K. pneumoniae isolated from pediatric patients. Therefore, it is important to monitor the spread of PMQR genes in clinical isolates to ensure efficient antibiotic use in those children. The finding denotes the importance of an antibiotics surveillance program.

the presence of comorbidities, and the history of antibiotic exposure.The preferred method of antibiotic administration is intravenously or intramuscularly 5 .
The common pathogen associated with healthcare sepsis is Gram-negative bacilli [6][7][8] .These pathogens develop antimicrobial resistance rapidly after misuse of antibiotics, lack of adherence to infection control measures, and the capacity of the organisms to develop antibiotic resistance through a wide range of mechanisms [9][10][11] .Antibiotic resistance leads to increased hospital costs with poor clinical outcomes 6 .
Escherichia coli (E.coli) and Klebsiella pneumoniae (K.pneumoniae) had an important role in antibiotic resistance due to the carriage of antibiotics resistance genetic determinant regions and their transport to other Enterobacteriaceae species 12,13 .The transfer of antibiotics resistance genetic determinant regions is mediated via plasmids and horizontal gene transfer 14 .In different countries, the resistance to antibiotics of E. coli and K. pneumoniae has been reported in the range of 85%-90% by the World Health Organization 15 .
Fluoroquinolone antibiotics like ciprofloxacin are broad antibiotics used effectively in the eradication of different bacterial pathogens 16 .However, with the emergence of resistance to this antibiotic, there is a reduction in therapeutic options for E. coli Klebsiella pneumoniae and other Enterobacteriaceae species 17 .Some guidelines approve the use of quinolones in pediatrics when the first line of treatment is not effective and no alternative is available 18,19 .Normally, the quinolone of choice is ciprofloxacin, due to its higher safety profile in comparison with other quinolones 20 .There are two mechanisms for the development of quinolone resistance, either through chromosomal mutation or through plasmid-mediated quinolone resistance (PMQR).Chromosomal mutations lead to the alteration of target enzymes and the affinity of drugs binding to them.The chromosomal mutations occur in the genes in the quinolone resistance determining regions (QRDRs).Plasmid-mediated quinolone resistance is mediated through the DNA gyrase protection and topoisomerase IV from the activity of quinolone antibiotics through the qnr genes namely, qnrA, qnrB, qnrC, qnrD, and qnrVC 21 .There is also involvement of the aminoglycoside-modifying enzyme through the gene aac(6')-Ib-cr by the acetylation of fluoroquinolones which leads to reduced susceptibility to quinolone antibiotics 21 .The other mechanism of quinolone resistance is the enhancement of the efflux pump activity facilitated by quinolone efflux pump (qepA) and oqxAB leading to the reduction in the susceptibility to quinolones and increased extended-spectrum beta-lactamase activity 22 .
The qnr genes lead to ciprofloxacin resistance by encoding proteins protecting DNA gyrase and topoisomerase IV from inhibition by quinolones 23 .The action of PMQR mutations is through the involvement of a modified enzyme that adds acetyl group C7 piperazine ring found in quinolones such as ciprofloxacin.This modification leads to the reduction of the drug's activity and confers resistance to quinolones 23 .
There are few reports about the emergence of quinolone resistance in E. coli and K. pneumoniae from pediatric patients to quinolone 24,25 .However, there are limited data about the resistance of Klebsiella pneumoniae and E. coli to ciprofloxacin from children with sepsis in Egypt.
Therefore, the present study aimed to study the presence of PMQR genes qnrA, qnrB, and qnrS by polymerase chain reaction (PCR) in E. coli and K. pneumoniae isolated from children with sepsis.

Method
This was a retrospective cross-sectional study including pediatric patients with healthcare-associated sepsis recruited from Mansoura University Children's Hospital, Egypt from January 2020 to January 2023.The included children were complaining of sepsis according to the criteria of the Center for Disease Control 7 with isolated bacterial pathogens Escherichia coli and Klebsiella pneumoniae by microbiological laboratory culture.Children with community-acquired sepsis or children with other pathogens associated with sepsis by microbiological culture were excluded from the study.The study was approved by the Mansoura Faculty of Medicine ethical committee (R.22.10.2354) and written approval was obtained from the parents of each child.

Microbiological culture
Five -milliliter blood samples were taken from each child and inoculated to the bottles of the blood culture system Bact/alert ® 3D (bioMérieux-France).Blood culture bottles found positive were subculture on MacConkey agar plates and blood agar plates at 37 °C for 24 h.Colonies were subjected to identification by Gram stain and further identification by Vitek 2 system (bioMérieux-France).Identified isolates K. pneumoniae and Escherichia coli were subjected to further studies for antibiotics sensitivity by disc diffusion method and molecular study by PCR for detection of PMQR genes.

Detection of resistance for ciprofloxacin by Broth Microdilution method (BMD)
E. coli and K. pneumoniae resistant to ciprofloxacin antibiotic disc were then tested for ciprofloxacin resistance by BMD method following the instruction of CLSI,2018 26 to identify the minimal inhibitory concentration (MIC).The isolates were suspended in 0.1 ml of Muller-Hinton broth and serial preparations of ciprofloxacin from 0.25 µg/ml to 128 µg/ml were added and incubation was performed at 37 °C for 20 h.Pure bacterial suspension in broth was used as positive control without adding ciprofloxacin.The well with the highest dilution with no turbidity was determined as MIC and the wells were sub-cultured to determine the minimal bactericidal concentration 17 .The interpretation was according to CLSI guidelines 17 at the following concentrations,

PCR for detection of PMQR genes qnrA, qnrB, and plasmid DNA extraction
Plasmid DNA purification was done using a commercial kit for plasmid DNA purification NucleoSpin ® Plasmid EasyPure (item 740727.50-Macherey-Nagel-Germany).The method was used according to the manufacturer's instructions.The extracted plasmid DNA was kept frozen at -20 °C till further amplification by PCR.

PCR
The used primers for the amplification of qnrA, qnrB, and qnrS genes were summarized in Table 1.The amplification mixture was a ready-to-use reagent mixture DreamTaq Green PCR Master Mix (Thermofisher Scientific, USA).
Amplification procedures were performed with the following conditions: initial denaturation at 94 °C for 5 min, followed by 35 cycles of denaturation at 94 °C for 30 s, (different annealing temperatures according to Table 1) for 30 s with an extension at 72 °C for 50 s, and a final extension for one cycle at 72 °C for 5 min in thermal cycler (Gene Amp PCR System 9700/Perkin Elmer Corporation, Norwal CT/USA) 27 .

Statistical analysis
The results of the study were analyzed by the SPPS22.0.Qualitative data was expressed as numbers and percentages and these data were compared using Chi-square and P was considered significant if < 0.05.The age was expressed as median, minimum, and maximum values as it was nonparametric.

Ethics approval and consent to participate
All methods were performed by the ethical standards as laid down in the Declaration of Helsinki and its later amendments or comparable ethical standards.The ethical approval of the study was obtained from the ethical committee of Mansoura Faculty of Medicine (R.22.10.2354) and written Informed consent was obtained from the parent of each child.

Results
The study included 52 (48.6%) males and 57(51.4%)females with ages ranging from two years to 15 years, median 8.00 years.Renal disorders (42.1%) followed by Diabetes mellitus (32.7%) were associated with sepsis.The associated devices with sepsis were mainly urinary catheters (37.4%) followed by peripheral venous catheter (34.6%) and central venous catheters (28.03%),Mean ± SD days of stay in ICU was 6.34 ± 2.15 days.The outcome of the sepsis was recovery in 97 patients (90.7%) and death in 10 patients (9.3%),Table 2.All children received empirical therapy of aminoglycosides plus beta-lactams antibiotics, data not shown.
Thirty-nine isolates were resistant to ciprofloxacin by disc diffusion method with 32 (82.1%) isolates being resistant to ciprofloxacin by MIC method.All the isolates resistant to ciprofloxacin by MIC were positive for PMQR genes by PCR, (Table 4).
The clinical isolates were E. coli from 52 of pediatric patients with sepsis and 55 K. pneumoniae from pediatric patients with sepsis.The most frequently detected qnr gene in Escherichia coli and Klebsiella pneumoniae was qnrA (28.8%, and 16.3% respectively).There was an insignificant difference between the frequency of positive PCR between E. coli and K. pneumonia (P = 0.11), and an insignificant difference in the frequency of qnrA, qnrB, and qnrS genes (P = 0.09, P = 0.54, P = 0.33 respectively), (Table 6).
The MDR resistance and the presence of qnr genes had insignificant associations (P = 0.53), but data was not shown.
The study of association between demographic data and clinical data with resistant isolates for ciprofloxacin by MIC revealed insignificant association between male and female (P = 0.86), age (P = 0.33), insignificant association between the presence of different comorbidities and resistant isolates (P = 0.98), insignificant association Table 1.The primers used with their sequences, the size of the amplified product base pair (bp), and the annealing temperature.www.nature.com/scientificreports/ between the presence of urinary catheter, central venous catheter and peripheral venous catheter (P = 0.87) and insignificant association between ICU stay days and the presence of resistant isolates (P = 0.4).There was an insignificant association between the outcome of sepsis and the presence of resistant isolates (P = 0.1970.There was a significant association between PCR and resistance to ciprofloxacin by MIC all isolates positive by PCR were resistant by MIC (P = 0.001),

Discussion
Pediatric patients admitted to hospitals are susceptible to healthcare-associated infections due to immature immune systems 28 .Bloodstream infections are among the most common infections 29 .
In the present study, E. coli and K. peumoniae were common Gram-Negative bacilli isolated from pediatric patients.This finding was reported previously [28][29][30][31] .There were medical devices associated with infections in those children such as peripheral venous catheters, urinary catheters, and central venous catheters.This finding indicates the necessity of proper implementation of infection control guidelines and early removal of medical devices to ensure minimal healthcare-associated infections 30 .
The children in the present study were complaining mainly of Diabetes mellitus and renal disorders as underlying comorbidities.The admission etiologies in developing countries are associated with medical conditions rather than surgical conditions in patients with young age 32 .The isolated E. coli and K. pneumoniae had high resistance to gentamicin (50.5%), levofloxacin (43.9%), amoxicillin (40.2%), and ciprofloxacin (36.4%).The results were in line with previous studies with high resistance to amoxicillin and ciprofloxacin 31,33 .The aminoglycosides such as gentamycin are usually used as the second choice combined with penicillin for the treatment of severe hospital-acquired infections as sepsis.Thus, this prescription can be responsible for the high resistance to gentamicin reported in the present study and previously 34 .The MDR was detected in 44.8% of the total isolates.This high percentage of multiple antibiotics resistance is a common finding worldwide among Gram-negative bacilli associated with healthcare-associated infections 31 .
Most resistant isolates to ciprofloxacin by disc diffusion method were resistant to ciprofloxacin by MIC method.The results of the disc diffusion method agree with MIC in the detection of resistant isolates to ciprofloxacin 35 .
Ciprofloxacin-resistant K. pneumoniae was (43.6%) and ciprofloxacin-resistant E. coli was (28.8%).A higher rate of resistant E. coli (64.4%) to ciprofloxacin was reported in a previous study 36 .The resistance rates differ according to the difference in the geographical regions due to the difference in the prescription of ciprofloxacin and due to the difference in the laboratory methods used in the reporting of the resistance 37 .
In the present study, all the resistant isolates for ciprofloxacin were positive for the PMQR genes.In a previous study, 80% of resistant isolates of E. coli and Klebsiella pneumoniae were positive for PMQR genes 38 .The high ciprofloxacin resistance among the isolates may be attributed to the selection mediated by other antibiotics such as cephalosporins leading to the selection of isolates carrying PMQR genes as previously reported 39 .In the present study, all isolates resistant to ciprofloxacin by MIC method were positive by PCR for PMQR genes, this finding supports the usefulness of the phenotypic resistant isolates to ciprofloxacin by MIC as a screening method due to its relatively low cost compared to molecular techniques.
The most frequent gene detected by PCR was qnrA (75%), followed by qnrB (28.1%), and qnrS (25%).On contrary to previous studies reporting that the prevalence of qnr genes ranged from 2% up to 40% in previous studies 40,41,38,42 .In Egypt, the rate of positive qnr genes in clinical isolates was 56.8% 43 .However, this difference in the rates of qnr gene detection may be attributed to the geographical difference, the difference in the type of infections of the studied population and their ages, and the difference in the prescription of antibiotics system.
The most prevalent gene was qnrA in both E. coli and K. pneumoniae.This finding was like the previous report including samples from blood culture specimens 44 On the contrary previous study reported that the qnrB gene was predominant in K. pneumoniae isolates 42 .
All qnr genes were present in two isolates (6.2%), the presence of qnrA and qnrB genes was detected in three isolates (9.4%), and qnrA plus qnrS was detected in two isolates.The compound presence of PMQR genes leads to the selection of resistant mutants 45 .
Ciprofloxacin can be used combined with beta-lactams antibiotics as a second line of therapy in sepsis if there was limited therapeutic response to the combined aminoglycosides with beta-lactams 46 .
The limitation of the study includes the need for more investigations including different resistance genes or virulence factor genes by different laboratory methods.

Conclusion
There was a high frequency of ciprofloxacin resistance among Escherichia coli and Klebsiella pneumoniae isolated from pediatric patients with healthcare-associated sepsis.There was a high prevalence of PMQR genes among E. coli and K. pneumoniae isolated from pediatric patients.Therefore, it is important to monitor the spread of PMQR genes in clinical isolates to ensure efficient antibiotic use in those children.The finding denotes the importance of an antibiotics surveillance program.

Table 7 Table 2 .
Basic demographic and clinical data of the studied children.

Table 3 .
Antibiotic resistance of the isolated E.coli versus K. pneumoniae.

Table 4 .
Phenotypic resistant isolates to ciprofloxacin and genotypic resistance by PCR.

Table 6 .
Frequency of PMQR genes in E.coli versus K. pneumonia.Significants values are in italic.

Table 7 .
Association between demographic, clinical data and PCR with resistant isolates to ciprofloxacin by MIC.